U.S. patent application number 13/172412 was filed with the patent office on 2012-02-02 for lrrk2 polynucleotides and transgenic animals.
Invention is credited to Jan O. Aasly, Matthew J. Farrer, Zbigniew K. Wszolek.
Application Number | 20120030781 13/172412 |
Document ID | / |
Family ID | 35276962 |
Filed Date | 2012-02-02 |
United States Patent
Application |
20120030781 |
Kind Code |
A1 |
Aasly; Jan O. ; et
al. |
February 2, 2012 |
LRRK2 POLYNUCLEOTIDES AND TRANSGENIC ANIMALS
Abstract
A polynucleotide consisting of the base sequence of SEQ ID NO:2,
or a complementary strand thereto, wherein the X is one of the
group being defined by the bases A, C or T. A primer and a probe
specific for that polynucleotide, wherein the primer and/or probe
contains at least 10 consecutive nucleotides, and finally use of
the probe for proving parkinsonism inheritance.
Inventors: |
Aasly; Jan O.; (Trondheim,
NO) ; Wszolek; Zbigniew K.; (Jacksonville, FL)
; Farrer; Matthew J.; (Jacksonville, FL) |
Family ID: |
35276962 |
Appl. No.: |
13/172412 |
Filed: |
June 29, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12433385 |
Apr 30, 2009 |
7993841 |
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13172412 |
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10568414 |
Jul 12, 2006 |
7544786 |
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PCT/NO05/00465 |
Dec 19, 2005 |
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12433385 |
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Current U.S.
Class: |
800/13 ;
435/320.1; 800/21 |
Current CPC
Class: |
C12Q 1/6883 20130101;
C12Q 2600/172 20130101; C12N 9/1205 20130101; C12Q 2600/112
20130101; A61P 43/00 20180101; C12Q 2600/156 20130101; A01K 67/0275
20130101 |
Class at
Publication: |
800/13 ;
435/320.1; 800/21 |
International
Class: |
A01K 67/027 20060101
A01K067/027; C12N 15/85 20060101 C12N015/85; C12N 15/63 20060101
C12N015/63 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 23, 2004 |
NO |
20045612 |
May 27, 2005 |
NO |
20052535 |
Claims
1. A method for transfecting an organism, comprising introducing
into a cell of the organism: (a) a polynucleotide comprising the
nucleotide sequence of SEQ ID NO:2, or a complementary strand
thereto, wherein the nucleotide at position 6055 of SEQ ID NO:2 is
A, C or T; or (b) a recombinant vector comprising the
polynucleotide.
2. The method of claim 1, wherein the organism is a non-human
mammal.
3. A recombinant vector comprising the nucleotide sequence of SEQ
ID NO:2, wherein the nucleotide at position 6055 of SEQ ID NO:2 is
A, C or T.
4. A transgenic non-human animal having integrated within its
genome a nucleic acid comprising the nucleotide sequence of SEQ ID
NO:2, wherein the nucleotide at position 6055 of SEQ ID NO:2 is A,
C, or T.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
12/433,385, filed Apr. 30, 2009, which is a continuation of U.S.
Ser. No. 10/568,414, filed Jul. 12, 2006, now U.S. Pat. No.
7,544,786, which is a National Stage application under 35 U.S.C.
.sctn.371 of International Application No. PCT/NO2005/00465, having
an International Filing Date of Dec. 19, 2005, which claims
priority from Norwegian Application No. 20052535, filed May 27,
2005, and Norwegian Application No. 20045612, filed on Dec. 23,
2004.
TECHNICAL FIELD
[0002] Present invention relates to a novel polynucleotide involved
in heritable Parkinson's disease (PD), a novel polypeptide encoded
by the polynucleotide, and a method for diagnosing heritable
Parkinson's disease (PD).
BACKGROUND
[0003] Parkinsonism (MIM168600) is a clinical syndrome
characterized by bradykinesia, resting tremor, muscle rigidity, and
postural instability (Gelb et al. 1999). The most common cause of
parkinsonism is Parkinson's disease (PD). Second to Alzheimer's
disease, PD is the most common neurodegenerative disorder affecting
>1% of the population over 55 years of age (de Rijk et al.
1995). Neuropathological findings in PD are loss of pigmented
neurons in the brainstem, substantia nigra and locus ceruleus, with
intracellular Lewy body inclusions found within surviving neurons
(Formo 1996).
[0004] Although PD is considered a sporadic disease, various
hereditary forms of parkinsonism have been recognized (Vila and
Przedborski 2004). A major breakthrough in recent years has been
the mapping and cloning of a number of genes causing monogenic
forms of parkinsonism. Genomic multiplication and missense
mutations in the .alpha.-synuclein gene were initially identified
in a small number of families with autosomal dominant parkinsonism
(PARK1/4 [MIM 168601]) (Polymeropoulos et al. 1997; Kruger et al.
1998; Singleton et al. 2003; Chartier-Harlin et al. 2004; Farrer et
al. 2004; Zarranz et al. 2004). Subsequently, .alpha.-synuclein
antibodies were found to robustly stain Lewy bodies and Lewy
neurites in the substantia nigra in familial and sporadic PD
(Spillantini et al. 1997) and common genetic variability in the
.alpha.-synuclein promoter has been implicated in sporadic PD (Pals
et al. 2004).
[0005] Autosomal recessive mutations in three genes, parkin, DJ-1
and PINK1 have been linked with early-onset parkinsonism (<45
years at onset) (PARK2, PARK6 & PARK7 [MIM 602533, 602544 &
608309]) (Kitada et al. 1998; Bonifati et al. 2003; Valente et al.
2004). A large number of pathogenic mutations and rearrangements
have been identified in the parkin gene reviewed by (Mata et al.
2004), but mutations in DJ-1 and PINK-1 arc rare (unpublished
data).
[0006] Very recently, five pathogenic mutations were identified in
a gene, leucine-rich repeat kinase 2 (LRRK2) in six families with
autosomal-dominant parkinsonism, linked to the PARK8 locus [MIM
607060]) (Zimprich et al. 2004a). Paisan-Ruiz and colleagues
independently confirmed these findings of two pathogenic mutations
in a British and Basque families (Paisan-Ruiz et al. x2004).
OBJECT
[0007] The object of the invention is to isolate a gene or
polynucleotide proving inheritable parkinsonism, and to use
presence of this gene to diagnose a patient before he/her gets
sick. A further object is to use this gene or polynucleotide to
transfect a microorganism or experimental animal in order to
develop a new medicine for treating or preventing the onset of
parkinsonism.
THE INVENTION
[0008] Inheritable parkinsonism may be proved by the method
according to the characterizing part of claim 5, and the other
objects are met by a polynucleotide according to the characterizing
part of claim 1, a recombinant vector according to claim 3, a DNA
probe and a DNA primer according to claims 4 and 6 respectively,
and a peptide according to claim 9.
[0009] The inventors have isolated a novel LRRK2 mutation, and this
mutation may cause development of dominantly inherited PD. By
screening healthy persons, one can state whether the healthy
persons have the mutation, and thus most likely will develop the
illness.
[0010] Using a probe to test whether a patient has the mutation
allows a precise, differential diagnosis of this type of
Parkinson's disease. The probe represents a safe and accurate
biomarker which will be powerful as it nominates subjects, future
patients, for neuroprotective therapy. At the present time this is
a research enterprise, but not for long. These subjects provide the
first (and only) `uniform substrate/background` for studies on drug
efficacy/safety. From a research perspective they will also
facilitate models of disease (C. elegans, Drosophila, mice) and
epidemiological research on the variable expressivity and
age-associated penetrance. As the sequence of the mutated gene is
known, microorganisms and further experimental animals may be
transfected, in order to investigate for a new medicine to treat or
prevent the onset of the illness.
[0011] The genetic information provides subjects with the cause of
their disease, an explanation for which, if handled correctly, can
be of great psychological benefit (fulfilling the `need to know`
why). This information also prioritizes the resources of the
research community, grant funding agencies and the pharmaceutical
industry on developing neuroprotective therapies to halt G2019S
disease progression.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] In the following the invention will be described by
reference to a study of PD patients and their families. Parts of
the study are shown in figures, wherein
[0013] FIG. 1 shows a schematic drawing of LRRK2 with predicted
protein domains. The LRRK2 protein sequence in the region of the
G2019S mutation is aligned for orthologs from human, rat, mouse,
and frog (all SEQ ID NO:24), as well as puffer fish (SEQ ID
NO:25).
[0014] FIG. 2 shows pedigrees of families with LRRK2 G2019S.
[0015] FIG. 3 shows chromosome 12q12 STR markers on the disease
haplotype (PARK 8).
[0016] FIG. 4 shows probability of becoming affected by
parkinsonism, in LRRK2 G2019S carriers, as a function of age.
[0017] FIG. 5 shows aligned amino acid sequences of the activation
loop of different human kinases: LRRK2 (SEQ ID NO:17), LRRK1 (SEQ
ID NO:18), MATK (SEQ ID NO:19), PDGFRA (SEQ ID NO:20), MAP3K10 (SEQ
ID NO:21), DAPK1 (SEQ ID NO:22), and BRAF (SEQ ID NO:23).
DETAILED DESCRIPTION
[0018] The inventors identified seven unrelated persons all having
the new mutation, from 248 multiplex kindreds with dominantly
inherited PD, and six further unrelated persons from three
population-based series of persons with dominantly inherited PD.
These 13 persons and their families made basis for the inventors'
further work. Segregation and linkage analysis provides evidence
for pathogenicity and an estimate of age-associated penetrance;
haplotype analysis demonstrates the mutation originates from a
common and ancient founder.
Subjects and Methods
Study Subjects
[0019] The patients and controls were examined by neurologists
specialized in movement disorders. A full history, including family
history and neurological examination, was completed on each
patient. Clinical diagnosis of PD required the presence of at least
two of three cardinal signs (resting tremor, bradykinesia and
rigidity), improvement from adequate dopaminergic therapy and the
absence of atypical features or other causes of parkinsonism.
LRRK2 Sequencing and Mutation Screening
[0020] Blood samples were taken and genomic DNA was extracted using
standard techniques. Six families (families 194, 281, 3081, 3082,
3083 and 3211) were known to have a positive LOD-score for STR
(Short Tandem Repeat) markers in the PARK8 locus (Zimprich et al.
2004b). Amplification of all 51 exons of the LRRK2 gene was
performed by polymerase chain reaction (PCR) in one patient having
PD, from each of these six families. All PCRs were carried out for
each primer set with 20-50 ng of template DNA in a total volume of
25 .mu.l using a final reaction concentration of 200 .mu.M dNTP,
1.times.PCR-Buffer (Qiagen), 1.times. Q-Solution (Qiagen), and 0.8
.mu.M of each primer. One unit of Taq polymerase (Qiagen) was added
to each reaction. Amplification was performed using a 57-52
C..degree.-touchdown protocol over 38 cycles. The primers used for
PCR amplification of LRRK2 exons and for sequencing are available
on request.
[0021] The nucleotide sequences of all PCR products were determined
by direct sequencing. Each PCR product was cleaned by using a
Millipore PCR purification plate. Three microliters of purified PCR
product was used per sequencing reaction with 1 .mu.l of either the
forward or reverse PCR primer and 1 .mu.l of BigDye reaction mix
(Applied Biosystems). Electrophoresis was performed under standard
conditions on an ABI 3730 automated sequencer (Applied Biosystems).
All sequences were obtained with both forward and reverse primers.
Sequences were analyzed with SeqScape software version 2.1.1
(Applied Biosystems) and compared with published sequence of LRRK2
(GenBank accession no. AY792511).
[0022] After identification of a heterozygous G2019S (G6055A)
mutation in the proband of family 3215 (referred to as family 3211
in Zimprich et al, 2004b), we designed a probe employing TaqMan
chemistry on an ABI7900 (Applied Biosystems) to screen for this
mutation. First we examined 248 PD patients from families with a
known family history, consistent with autosomal dominant
transmission of a suspected causative gene. Then 377 Norwegian, 271
Irish and 100 Polish PD patients (constituting the three population
series) were checked using this assay; and 2260 samples of healthy
persons from similar populations were also included (1200 US
American, 550 Norwegian, 330 Irish and 180 Polish subjects), the
latter to be used as control samples. Mutations were confirmed by
direct sequencing of PCR products from LRRK2 exon 41. Finally, all
participating family members of LRRK2 G2019 mutation carriers
(affected and unaffected) were screened for the mutation.
[0023] By 6055 G>A or G6055A it is meant that nucleotide number
6055 of the LRRK2 gene, counted from the 5'end of the
polynucleotide, has changed from G (guanine) to A (adenine). This
change also causes a change in the polypeptide encoded by the
polynucleotide, and G2019S denotes a polynucleotide where amino
acid number 2019 is changed from G (Glycine) to S (Serine). These
shortenings are well known to persons skilled of the art.
Genotyping of STR Markers
[0024] Fourteen STR markers were genotyped in mutation carriers and
all available family members, in all 13 families, for linkage
analyses and to determine whether there was a particular haplotype
associated with the LRRK2 mutation. STR markers were chosen to span
the PARK8 region including D12S87, D12S1648, D12S2080, D12S2194,
D12S1048, D12S1301 and D12S1701. LRRK2 is located between D12S2194
and D12S1048. We also developed seven novel STR markers in this
region (shown in table 1 below) by searching for repeat
polymorphisms using RepeatMasker of in silico BAC sequence (UCSC
Human Genome Browser Web site). The labeling of these novel markers
reflects their physical position relative to the start codon of
LRRK2.
TABLE-US-00001 TABLE 1 Novel chromosome 12 STR markers Physical
position Marker (bp) On name Primer sequence chromosome 12 SEQ ID
NO: D12S2514 F: 5'-TTGCAGCTGTAAGGAATTTGGG-3' 38873779 3 R:
5'-GCATTCTTCAGCCTGAGACCC-3' 4 D12S2515 F:
5'-TGAAGGACACTGAACAAGATGG-3' 38974140 5 R:
5'-GCCATAGTCCTTCCATAGTTCC-3' 6 D12S2516 F: 5'-CGCAGCGAGCATTGTACC-3'
38989214 7 R: 5'-CTCGGAAAGTTTCCCAATTC-3' 8 D12S2518 F:
5'-CTGGTATTACCTCAACTGTGGCTC-3' 39034800 9 R:
5'-ACTGGTATGTTTAAGCCTGGCAC-3' 10 D12S2519 F:
5'-AGCAGCAGAGAAGATTTCAATAAC-3' 39116816 11 R:
5'-AATCATCTTTGAAAGAACCAGG-3' 12 D12S2523 F:
5'-TAAACGAAGCTCCCTCACTGTAAG-3' 39147728 13 R:
5'-TCTTTGTAGCTGCGGTTGTTTC-3' 14 D12S2517 F:
5'-TCATGAAGATGTCTGTGATAGGGC-3' 39282976 15 R:
5'-CTCTATTGTGAGCAAACTGCATGG-3' 16
[0025] One primer of each pair was labeled with a fluorescent tag.
PCR reactions were carried out on 10-20 ng of DNA in a total volume
of 15 .mu.l with final reaction concentrations of 150 .mu.M dNTP,
1.times.PCR-Buffer (Qiagen), 1.times. Q-Solution (Qiagen) and 0.6
.mu.M of each primer, with 1 unit of Taq Polymerase (Qiagen).
Amplification was performed using a 57-52.degree. C.-touchdown
protocol over 38 cycles. The PCR product for each marker was
diluted by a factor of 10 to 100 with water. One microliter was
then added to 10 .mu.l of Hi-Di Formamide and Rox size standard.
All samples were run on an ABI 3100 genetic analyzer, and results
were analyzed using Genescan 3.7 and Genotyper 3.7 software
(Applied Biosystems). Since population allele frequencies were not
available from the CEPH database, these have been estimated by
genotyping 95 unrelated Caucasian subjects, a population based
series from the United States (shown in table 2 below).
TABLE-US-00002 TABLE 2 Allele frequencies of Park 8 Markers Marker
and allele (bp) Frequency (%) D12S87 (n = 92) 150 0.5 154 1.1 156
27.2 158 33.2 160 11.4 162 2.7 164 6.0 166 17.4 168 0.5 D12S1648 (n
= 91) 110 13.7 112 3.3 114 11.0 116 4.4 118 2.2 120 2.8 122 17.0
124 3.9 126 7.7 128 14.3 130 8.8 132 2.8 134 2.8 136 1.7 138 0.6
140 2.2 142 1.1 D12S2080 (n = 93) 176 1.6 180 20.2 184 44.7 188
22.9 192 10.6 D12S2194 (n = 87) 245 0.6 249 40.9 253 32.4 257 19.9
261 4.6 265 1.7 D12S2514 (n = 82) 284 11.0 291 53.1 294 32.3 297
1.2 300 2.4 D12S2515 (n = 93) 208 3.2 212 26.6 216 18.6 220 22.9
224 20.7 228 5.3 232 2.7 rs 7966550 (n = 90) T 90.6 C 9.4 DS12S2516
252 37.3 254 62.7 rs 1427263 (n = 89) A 63.6 C 36.5 rs1116013 (n =
88) A 49.4 G 50.6 rs11564148 (n = 88) A 26.1 T 73.9 D12S2518 (N =
90) 154 79.7 168 15.9 170 4.4 D12S519 (n = 72) 132 29.5 134 22.6
138 22.6 140 25.3 D12S2520 (N = 85) 248 8.2 251 7.6 254 10.0 257
54.1 260 20.0 D12S2521 (N = 93) 311 0.5 315 10.8 319 20.4 323 8.1
327 7.0 331 8.1 335 0.5 355 1.1 359 7.5 363 13.4 367 7.0 371 7.0
375 6.5 379 3.8 383 1.1 387 .5 D12S2522 (N = 93) 281 9.1 283 14.0
285 .5 287 11.3 293 .5 295 15.6 297 44.6 299 4.3 D12S2523 (n = 89)
305 18.9 314 41.1 317 8.9 320 30.0 323 1.1 180 8.5 182 7.5 184 15.4
186 8.5 188 11.7 190 8.0 192 5.3 194 1.1 196 1.1 198 3.2 200 0.5
202 3.7 204 6.9 206 6.9 208 4.3 210 2.1 212 3.2 214 1.6 216 0.5
D12S1048 (n = 89) 211 37.2 214 21.1 217 17.8 220 2.2 223 6.7 226
11.7 229 3.3 D12S1301 (n = 93) 96 0.5 100 37.2 104 17.6 108 11.1
112 12.2 116 13.3 120 7.5 124 0.5 D12S1701 (n = 93) 89 4.3 91 4.8
93 10.8 95 40.0 97 16.0 99 12.4 101 11.8 103 0.5 A The number of
individuals genotyped is given for each marker (n) B Alle
frequencies are for individual markers in U.S. control subjects
Statistical Analysis
[0026] Multipoint nonparametric LOD scores for all families were
calculated using GENEHUNTER-PLUS (Kong and Cox 1997). The frequency
of the deleterious allele was set at 0.0001, and empirically
determined allele frequencies were employed. The map positions for
each marker were taken from Rutgers combined linkage-physical map
version 1.0 (MAP-O-MAT web site). The three loci D12S2080, D12S2194
and D12S1301 are very tightly linked, with no observed recombinants
in the database or within our genotyped families, and thus
inter-marker distances were assigned as 0.01 cM.
[0027] Chromosome 12 haplotypes in the PARK8 region were
established for those families in which chromosome phase for
mutation-carrying individuals could be deduced, thereby determining
which alleles co-segregated with the LRRK2 G2019S mutation in each
family. For those affected individuals in whom the associated
allele for a marker could not be determined, both alleles are
given.
[0028] The age-dependent penetrance was estimated as the
probability of a gene carrier becoming affected, at a given age,
within the 13 families. The number of affected mutation carriers,
for each decade, was divided by the total number of affected
individuals, plus the number of unaffected carriers within that
range. For some affected family members no DNA was available and
only historical data on the disease course was obtained. These
individuals were excluded from penetrance calculations.
Results
[0029] As mentioned previously, we identified 13 affected probands
(i.e. 13 patients) who carry a heterozygous G6055A mutation in exon
41 of the LRRK2 gene. The mutation leads to a G2019S amino acid
substitution of a highly conserved residue within the predicted
activation loop of the MAPKKK (Mitogen-Activated Protein Kinase
Kinase Kinase) domain (FIG. 1). After genotyping a total of 42
additional family members, 22 additional subjects were found to
carry the mutation, seven with a diagnosis of PD (shown in table 3
below). One affected member of family P-089 did not carry the
mutation and, for the purposes of this study, was considered a
phenocopy and excluded from further analyses. Seven families
originated from Norway, three were from the United States, two from
Ireland, and one was from Poland. One family from the United States
descended from Russian/Rumania, and another from Italy. For only
one family (family 111), the ethnic origin was unknown. The LRRK2
G2019S mutation segregates with disease in all kindreds, consistent
with autosomal dominant transmission. To ensure patient
confidentiality, simplified versions of the family pedigrees are
presented in FIG. 2. There was no evidence of the mutation in the
2260 control samples.
[0030] Age at onset of clinical symptoms was quite variable, even
within the same family. Family 1120, a family from the United
States, had both the earliest and latest age at onset for a
patient. The youngest affected subject had an onset at 39 years,
whereas the oldest carrier presented with initial symptoms at 78
years. Where recorded, most LRRK2 G2019S carriers have late-onset
disease (>50 years at onset). The mean age at onset of affected
mutation carriers was 56.8 years (range 39-78 years, n=19).
Unaffected carriers have a mean age of 53.9 years (range 26-74
years, n=14). The penetrance of the mutation was found to be highly
age-dependent, increasing from 17% at the age of 50 to 85% at the
age of 70 (FIG. 4).
TABLE-US-00003 TABLE 3 Demographic and Clinical Information for 13
Families with LRRK2 G2019S FINDINGS FOR FAMILY CHARACTERISTIC P-063
P-089 P-104 P-241 P-369 P-394 F05 1210 111 1120 PD66 3211 IP
Country of origin Norway Norway Norway Norway Norway Norway Norway
United United United Ireland Ireland Poland States States States
No. of generations 3 4 3 3 3 4 4 2 2 3 1 2 1 No. of affected 2 4 4
1 3 4 5 2 3 3 1 3 1 individuals No. of typed individuals 1(6) 2(8)
1(1) 1(4) 2(3) 1(1) 3(6) 1(0) 2(0) 3(3) 1(0) 2(6) 1(0) affected
(unaffected) No. of typed generations 2 3 1 2 1 2 2 1 1 2 1 1 1
Age.sup.3 at onset in years 59 59 58 60 50 66 64 65 58 59 41 46 73
(range) (53-65) (43-70) (43-61) (61-70) (57-58) (39-78) (40-52)
Maximum mLOD score 0 .30 0 0 .60 0 .90 0 .09 .30 0 .30 0
.sup.3Average ages at onset are given when affected individuals. n
.gtoreq. 2 Evidence for linkage (the statistical burden of proof
that this mutation causes disease) to the PARK8 locus was found
across families, with a combined maximum multipoint LOD score of
2.41 [for all 14 markers], corresponding to a P value of 4.3.
.times. 10.sup.-4 As only a defined chromosomal region was
investigated, rather than a genome-wide search, this LOD score
exceeds that required for significance, P = 0.01 (Lander and
Kruglyak 1995). A positive LOD score was found in all families
where more then one affected subject was genotyped (table 3).
[0031] All affected members from the different families, except the
individual in family P-089 who did not carry the mutation, appear
to share a common haplotype on chromosome 12 the LRRK2 gene locus
(FIG. 3). Haplotypes can be established with certainty in nine of
the families, and all mutation carriers in these families share
alleles for four STR markers and 4 single nucleotide polymorphisms
(SNPs) in the LRRK2 gene locus. These markers are LRRK2 D12S2516,
D12S2518, D12S2519, D12S2520 and SNPs rs7966550, rs1427263,
rs11176013, rs11564148. For the remaining families, the number of
available samples from relatives was not sufficient to determine
phase. However, the genotypes in these cases are consistent with a
common LRRK2 G2019S allele. D12S2516 is located in intron 29 and
D12S2518 is located in intron 44 of the LRRK2 gene, whereas the two
other shared markers are positioned 3' of the gene. Using the
physical position of the shared and non-shared markers, the size of
the shared haplotype is between 145 kb and 154 kb.
Discussion
[0032] We have identified a novel LRRK2 mutation, G2019S, which
co-segregates with autosomal dominant parkinsonism in 13 kindreds
originating from several European populations. Positive LOD scores
were obtained in multiplex families, and combined they provide
significant support for the PARK8 locus. LRRK2 G2019S mutation was
absent in a large number of control subjects, and of similar
ethnicity. The number of families linked to LRRK2 in this and
previous studies now explains the majority of genetically defined
autosomal dominant parkinsonism.
[0033] The mean age at onset of affected LRRK2 G2019S carriers was
56.8 years, and comparable to that of patients in other families
linked to PARK8 (Funayama et al. 2002; Paisan-Ruiz et al. 2004;
Zimprich et al. 2004a). The majority of patients present with
late-onset disease, indistinguishable from typical idiopathic PD.
Disease penetrance is age-dependent, and increases in a linear
fashion from 17% at the age of 50 to 85% at the age of 70. Age is
the single most consistent risk factor for development of PD and
other neurodegenerative disorders (Lang and Lozano 1998), and an
important risk factor in LRRK2 associated parkinsonism.
Interestingly, age at onset was variable in this study, both within
and between different families, suggesting other susceptibility
factors, environmental or genetic, may influence the phenotype.
[0034] Although our findings clearly indicate that LRRK2 mutations
account for a substantial proportion of familial late-onset
parkinsonism, historically, cross-sectional twin studies have not
supported a genetic etiology for late-onset PD (Tanner et al. 1999;
Wirdefeldt et al. 2004). The age-associated penetrance of LRRK2
mutations provides some explanation as even large and well designed
twin studies are underpowered to detect incompletely penetrant
mutations (Simon et al. 2002). LRRK2 mutations were also found in
apparently sporadic PD patients; three of the patients in this
study did not have any known affected first- or second-degree
relatives. However, a caveat of age-dependent penetrance is that
carriers may die of other diseases, before manifesting or being
diagnosed with PD. Thus, it seems difficult to separate sporadic
and familial PD, or to hypothesize environmental causes to be more
important in one group and genetic causes more prominent in the
other. In light of these results, a family history of parkinsonism,
previously considered an exclusion criterion for a diagnosis of PD,
must be reconsidered (Hughes et al. 1992).
[0035] LRRK2 is a member of the recently defined ROCO protein
family (Bosgraaf and Van Haastert 2003). In human, mouse and rat,
members of the ROCO protein family have five conserved domains
(FIG. 1). The kinase domain belongs to the MAPKKK subfamily of
kinases. The active sites of all kinases are located in a cleft
between an N-terminal and a C-terminal lobe, typically covered by
an `activation loop`, in an inactive conformation. The activation
loop must undergo crucial structural changes to allow access to
peptide substrates and to orientate key catalytic amino acids (Huse
and Kuriyan 2002). In different kinases, the activation loop starts
and ends with the conserved residues asp-phe-gly (DFG) and
ala-pro-glu (APE), respectively (Dibb et al. 2004). Of note, the
LRRK2 G2019S substitution changes a highly conserved amino acid at
the start of this loop (FIG. 5). In a German family we previously
described, an 12020T mutation is located in an adjacent codon
(Zimprich et al. 2004a). In other kinases, oncogenic mutations in
residues within the activation loop of the kinase domain have an
activating effect (Davies et al. 2002), thus we postulate LRRK2
G2019S and 12020T mutations may have an effect on its kinase
activity.
[0036] The age of an allele may be estimated from the genetic
variation among different copies (intra-allelic variation), or from
its frequency (Slatkin and Rannala 2000). However, the local
recombination rate on chromosome 12q12 is unknown, as is the
frequency of the G2019S mutation in the general population.
Nevertheless, at centromeres there is generally a dearth in
recombination; indeed no crossovers have been observed between
LRRK2 flanking markers D12S2194 and D12S1048 in our studies, or
within CEPH families (MAP-O-MAT web site). The physical size of the
shared haplotype is also small, between 145 kb and 154 kb, and the
allele is widespread in families from several European populations.
Hence, the mutation is likely to be ancient and may be relatively
common in specific populations. These data suggest a substantial
proportion of late-onset PD will have a genetic basis.
Electronic-Database Information
[0037] The physical position of markers is from NCBI build 34.
Accession numbers and URLs for data presented herein are as
follows:
Online Mendelian Inheritance in Man (OMIM), World Wide Web at
ncbi.nlm.nih.gov/Omim/MAP-O-MAT, compgen.rutgers.edu/mapomat
RepeatMasker, World Wide Web at repeatmasker.org/
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Sequence CWU 1
1
2512527PRTHomo sapiensVARIANT2019Xaa = Any Amino Acid 1Met Ala Ser
Gly Ser Cys Gln Gly Cys Glu Glu Asp Glu Glu Thr Leu1 5 10 15Lys Lys
Leu Ile Val Arg Leu Asn Asn Val Gln Glu Gly Lys Gln Ile 20 25 30Glu
Thr Leu Val Gln Ile Leu Glu Asp Leu Leu Val Phe Thr Tyr Ser 35 40
45Glu His Ala Ser Lys Leu Phe Gln Gly Lys Asn Ile His Val Pro Leu
50 55 60Leu Ile Val Leu Asp Ser Tyr Met Arg Val Ala Ser Val Gln Gln
Val65 70 75 80Gly Trp Ser Leu Leu Cys Lys Leu Ile Glu Val Cys Pro
Gly Thr Met 85 90 95Gln Ser Leu Met Gly Pro Gln Asp Val Gly Asn Asp
Trp Glu Val Leu 100 105 110Gly Val His Gln Leu Ile Leu Lys Met Leu
Thr Val His Asn Ala Ser 115 120 125Val Asn Leu Ser Val Ile Gly Leu
Lys Thr Leu Asp Leu Leu Leu Thr 130 135 140Ser Gly Lys Ile Thr Leu
Leu Ile Leu Asp Glu Glu Ser Asp Ile Phe145 150 155 160Met Leu Ile
Phe Asp Ala Met His Ser Phe Pro Ala Asn Asp Glu Val 165 170 175Gln
Lys Leu Gly Cys Lys Ala Leu His Val Leu Phe Glu Arg Val Ser 180 185
190Glu Glu Gln Leu Thr Glu Phe Val Glu Asn Lys Asp Tyr Met Ile Leu
195 200 205Leu Ser Ala Ser Thr Asn Phe Lys Asp Glu Glu Glu Ile Val
Leu His 210 215 220Val Leu His Cys Leu His Ser Leu Ala Ile Pro Cys
Asn Asn Val Glu225 230 235 240Val Leu Met Ser Gly Asn Val Arg Cys
Tyr Asn Ile Val Val Glu Ala 245 250 255Met Lys Ala Phe Pro Met Ser
Glu Arg Ile Gln Glu Val Ser Cys Cys 260 265 270Leu Leu His Arg Leu
Thr Leu Gly Asn Phe Phe Asn Ile Leu Val Leu 275 280 285Asn Glu Val
His Glu Phe Val Val Lys Ala Val Gln Gln Tyr Pro Glu 290 295 300Asn
Ala Ala Leu Gln Ile Ser Ala Leu Ser Cys Leu Ala Leu Leu Thr305 310
315 320Glu Thr Ile Phe Leu Asn Gln Asp Leu Glu Glu Lys Asn Glu Asn
Gln 325 330 335Glu Asn Asp Asp Glu Gly Glu Glu Asp Lys Leu Phe Trp
Leu Glu Ala 340 345 350Cys Tyr Lys Ala Leu Thr Trp His Arg Lys Asn
Lys His Val Gln Glu 355 360 365Ala Ala Cys Trp Ala Leu Asn Asn Leu
Leu Met Tyr Gln Asn Ser Leu 370 375 380His Glu Lys Ile Gly Asp Glu
Asp Gly His Phe Pro Ala His Arg Glu385 390 395 400Val Met Leu Ser
Met Leu Met His Ser Ser Ser Lys Glu Val Phe Gln 405 410 415Ala Ser
Ala Asn Ala Leu Ser Thr Leu Leu Glu Gln Asn Val Asn Phe 420 425
430Arg Lys Ile Leu Leu Ser Lys Gly Ile His Leu Asn Val Leu Glu Leu
435 440 445Met Gln Lys His Ile His Ser Pro Glu Val Ala Glu Ser Gly
Cys Lys 450 455 460Met Leu Asn His Leu Phe Glu Gly Ser Asn Thr Ser
Leu Asp Ile Met465 470 475 480Ala Ala Val Val Pro Lys Ile Leu Thr
Val Met Lys Arg His Glu Thr 485 490 495Ser Leu Pro Val Gln Leu Glu
Ala Leu Arg Ala Ile Leu His Phe Ile 500 505 510Val Pro Gly Met Pro
Glu Glu Ser Arg Glu Asp Thr Glu Phe His His 515 520 525Lys Leu Asn
Met Val Lys Lys Gln Cys Phe Lys Asn Asp Ile His Lys 530 535 540Leu
Val Leu Ala Ala Leu Asn Arg Phe Ile Gly Asn Pro Gly Ile Gln545 550
555 560Lys Cys Gly Leu Lys Val Ile Ser Ser Ile Val His Phe Pro Asp
Ala 565 570 575Leu Glu Met Leu Ser Leu Glu Gly Ala Met Asp Ser Val
Leu His Thr 580 585 590Leu Gln Met Tyr Pro Asp Asp Gln Glu Ile Gln
Cys Leu Gly Leu Ser 595 600 605Leu Ile Gly Tyr Leu Ile Thr Lys Lys
Asn Val Phe Ile Gly Thr Gly 610 615 620His Leu Leu Ala Lys Ile Leu
Val Ser Ser Leu Tyr Arg Phe Lys Asp625 630 635 640Val Ala Glu Ile
Gln Thr Lys Gly Phe Gln Thr Ile Leu Ala Ile Leu 645 650 655Lys Leu
Ser Ala Ser Phe Ser Lys Leu Leu Val His His Ser Phe Asp 660 665
670Leu Val Ile Phe His Gln Met Ser Ser Asn Ile Met Glu Gln Lys Asp
675 680 685Gln Gln Phe Leu Asn Leu Cys Cys Lys Cys Phe Ala Lys Val
Ala Met 690 695 700Asp Asp Tyr Leu Lys Asn Val Met Leu Glu Arg Ala
Cys Asp Gln Asn705 710 715 720Asn Ser Ile Met Val Glu Cys Leu Leu
Leu Leu Gly Ala Asp Ala Asn 725 730 735Gln Ala Lys Glu Gly Ser Ser
Leu Ile Cys Gln Val Cys Glu Lys Glu 740 745 750Ser Ser Pro Lys Leu
Val Glu Leu Leu Leu Asn Ser Gly Ser Arg Glu 755 760 765Gln Asp Val
Arg Lys Ala Leu Thr Ile Ser Ile Gly Lys Gly Asp Ser 770 775 780Gln
Ile Ile Ser Leu Leu Leu Arg Arg Leu Ala Leu Asp Val Ala Asn785 790
795 800Asn Ser Ile Cys Leu Gly Gly Phe Cys Ile Gly Lys Val Glu Pro
Ser 805 810 815Trp Leu Gly Pro Leu Phe Pro Asp Lys Thr Ser Asn Leu
Arg Lys Gln 820 825 830Thr Asn Ile Ala Ser Thr Leu Ala Arg Met Val
Ile Arg Tyr Gln Met 835 840 845Lys Ser Ala Val Glu Glu Gly Thr Ala
Ser Gly Ser Asp Gly Asn Phe 850 855 860Ser Glu Asp Val Leu Ser Lys
Phe Asp Glu Trp Thr Phe Ile Pro Asp865 870 875 880Ser Ser Met Asp
Ser Val Phe Ala Gln Ser Asp Asp Leu Asp Ser Glu 885 890 895Gly Ser
Glu Gly Ser Phe Leu Val Lys Lys Lys Ser Asn Ser Ile Ser 900 905
910Val Gly Glu Phe Tyr Arg Asp Ala Val Leu Gln Arg Cys Ser Pro Asn
915 920 925Leu Gln Arg His Ser Asn Ser Leu Gly Pro Ile Phe Asp His
Glu Asp 930 935 940Leu Leu Lys Arg Lys Arg Lys Ile Leu Ser Ser Asp
Asp Ser Leu Arg945 950 955 960Ser Ser Lys Leu Gln Ser His Met Arg
His Ser Asp Ser Ile Ser Ser 965 970 975Leu Ala Ser Glu Arg Glu Tyr
Ile Thr Ser Leu Asp Leu Ser Ala Asn 980 985 990Glu Leu Arg Asp Ile
Asp Ala Leu Ser Gln Lys Cys Cys Ile Ser Val 995 1000 1005His Leu
Glu His Leu Glu Lys Leu Glu Leu His Gln Asn Ala Leu Thr 1010 1015
1020Ser Phe Pro Gln Gln Leu Cys Glu Thr Leu Lys Ser Leu Thr His
Leu1025 1030 1035 1040Asp Leu His Ser Asn Lys Phe Thr Ser Phe Pro
Ser Tyr Leu Leu Lys 1045 1050 1055Met Ser Cys Ile Ala Asn Leu Asp
Val Ser Arg Asn Asp Ile Gly Pro 1060 1065 1070Ser Val Val Leu Asp
Pro Thr Val Lys Cys Pro Thr Leu Lys Gln Phe 1075 1080 1085Asn Leu
Ser Tyr Asn Gln Leu Ser Phe Val Pro Glu Asn Leu Thr Asp 1090 1095
1100Val Val Glu Lys Leu Glu Gln Leu Ile Leu Glu Gly Asn Lys Ile
Ser1105 1110 1115 1120Gly Ile Cys Ser Pro Leu Arg Leu Lys Glu Leu
Lys Ile Leu Asn Leu 1125 1130 1135Ser Lys Asn His Ile Ser Ser Leu
Ser Glu Asn Phe Leu Glu Ala Cys 1140 1145 1150Pro Lys Val Glu Ser
Phe Ser Ala Arg Met Asn Phe Leu Ala Ala Met 1155 1160 1165Pro Phe
Leu Pro Pro Ser Met Thr Ile Leu Lys Leu Ser Gln Asn Lys 1170 1175
1180Phe Ser Cys Ile Pro Glu Ala Ile Leu Asn Leu Pro His Leu Arg
Ser1185 1190 1195 1200Leu Asp Met Ser Ser Asn Asp Ile Gln Tyr Leu
Pro Gly Pro Ala His 1205 1210 1215Trp Lys Ser Leu Asn Leu Arg Glu
Leu Leu Phe Ser His Asn Gln Ile 1220 1225 1230Ser Ile Leu Asp Leu
Ser Glu Lys Ala Tyr Leu Trp Ser Arg Val Glu 1235 1240 1245Lys Leu
His Leu Ser His Asn Lys Leu Lys Glu Ile Pro Pro Glu Ile 1250 1255
1260Gly Cys Leu Glu Asn Leu Thr Ser Leu Asp Val Ser Tyr Asn Leu
Glu1265 1270 1275 1280Leu Arg Ser Phe Pro Asn Glu Met Gly Lys Leu
Ser Lys Ile Trp Asp 1285 1290 1295Leu Pro Leu Asp Glu Leu His Leu
Asn Phe Asp Phe Lys His Ile Gly 1300 1305 1310Cys Lys Ala Lys Asp
Ile Ile Arg Phe Leu Gln Gln Arg Leu Lys Lys 1315 1320 1325Ala Val
Pro Tyr Asn Arg Met Lys Leu Met Ile Val Gly Asn Thr Gly 1330 1335
1340Ser Gly Lys Thr Thr Leu Leu Gln Gln Leu Met Lys Thr Lys Lys
Ser1345 1350 1355 1360Asp Leu Gly Met Gln Ser Ala Thr Val Gly Ile
Asp Val Lys Asp Trp 1365 1370 1375Pro Ile Gln Ile Arg Asp Lys Arg
Lys Arg Asp Leu Val Leu Asn Val 1380 1385 1390Trp Asp Phe Ala Gly
Arg Glu Glu Phe Tyr Ser Thr His Pro His Phe 1395 1400 1405Met Thr
Gln Arg Ala Leu Tyr Leu Ala Val Tyr Asp Leu Ser Lys Gly 1410 1415
1420Gln Ala Glu Val Asp Ala Met Lys Pro Trp Leu Phe Asn Ile Lys
Ala1425 1430 1435 1440Arg Ala Ser Ser Ser Pro Val Ile Leu Val Gly
Thr His Leu Asp Val 1445 1450 1455Ser Asp Glu Lys Gln Arg Lys Ala
Cys Met Ser Lys Ile Thr Lys Glu 1460 1465 1470Leu Leu Asn Lys Arg
Gly Phe Pro Ala Ile Arg Asp Tyr His Phe Val 1475 1480 1485Asn Ala
Thr Glu Glu Ser Asp Ala Leu Ala Lys Leu Arg Lys Thr Ile 1490 1495
1500Ile Asn Glu Ser Leu Asn Phe Lys Ile Arg Asp Gln Leu Val Val
Gly1505 1510 1515 1520Gln Leu Ile Pro Asp Cys Tyr Val Glu Leu Glu
Lys Ile Ile Leu Ser 1525 1530 1535Glu Arg Lys Asn Val Pro Ile Glu
Phe Pro Val Ile Asp Arg Lys Arg 1540 1545 1550Leu Leu Gln Leu Val
Arg Glu Asn Gln Leu Gln Leu Asp Glu Asn Glu 1555 1560 1565Leu Pro
His Ala Val His Phe Leu Asn Glu Ser Gly Val Leu Leu His 1570 1575
1580Phe Gln Asp Pro Ala Leu Gln Leu Ser Asp Leu Tyr Phe Val Glu
Pro1585 1590 1595 1600Lys Trp Leu Cys Lys Ile Met Ala Gln Ile Leu
Thr Val Lys Val Glu 1605 1610 1615Gly Cys Pro Lys His Pro Lys Gly
Ile Ile Ser Arg Arg Asp Val Glu 1620 1625 1630Lys Phe Leu Ser Lys
Lys Arg Lys Phe Pro Lys Asn Tyr Met Ser Gln 1635 1640 1645Tyr Phe
Lys Leu Leu Glu Lys Phe Gln Ile Ala Leu Pro Ile Gly Glu 1650 1655
1660Glu Tyr Leu Leu Val Pro Ser Ser Leu Ser Asp His Arg Pro Val
Ile1665 1670 1675 1680Glu Leu Pro His Cys Glu Asn Ser Glu Ile Ile
Ile Arg Leu Tyr Glu 1685 1690 1695Met Pro Tyr Phe Pro Met Gly Phe
Trp Ser Arg Leu Ile Asn Arg Leu 1700 1705 1710Leu Glu Ile Ser Pro
Tyr Met Leu Ser Gly Arg Glu Arg Ala Leu Arg 1715 1720 1725Pro Asn
Arg Met Tyr Trp Arg Gln Gly Ile Tyr Leu Asn Trp Ser Pro 1730 1735
1740Glu Ala Tyr Cys Leu Val Gly Ser Glu Val Leu Asp Asn His Pro
Glu1745 1750 1755 1760Ser Phe Leu Lys Ile Thr Val Pro Ser Cys Arg
Lys Gly Cys Ile Leu 1765 1770 1775Leu Gly Gln Val Val Asp His Ile
Asp Ser Leu Met Glu Glu Trp Phe 1780 1785 1790Pro Gly Leu Leu Glu
Ile Asp Ile Cys Gly Glu Gly Glu Thr Leu Leu 1795 1800 1805Lys Lys
Trp Ala Leu Tyr Ser Phe Asn Asp Gly Glu Glu His Gln Lys 1810 1815
1820Ile Leu Leu Asp Asp Leu Met Lys Lys Ala Glu Glu Gly Asp Leu
Leu1825 1830 1835 1840Val Asn Pro Asp Gln Pro Arg Leu Thr Ile Pro
Ile Ser Gln Ile Ala 1845 1850 1855Pro Asp Leu Ile Leu Ala Asp Leu
Pro Arg Asn Ile Met Leu Asn Asn 1860 1865 1870Asp Glu Leu Glu Phe
Glu Gln Ala Pro Glu Phe Leu Leu Gly Asp Gly 1875 1880 1885Ser Phe
Gly Ser Val Tyr Arg Ala Ala Tyr Glu Gly Glu Glu Val Ala 1890 1895
1900Val Lys Ile Phe Asn Lys His Thr Ser Leu Arg Leu Leu Arg Gln
Glu1905 1910 1915 1920Leu Val Val Leu Cys His Leu His His Pro Ser
Leu Ile Ser Leu Leu 1925 1930 1935Ala Ala Gly Ile Arg Pro Arg Met
Leu Val Met Glu Leu Ala Ser Lys 1940 1945 1950Gly Ser Leu Asp Arg
Leu Leu Gln Gln Asp Lys Ala Ser Leu Thr Arg 1955 1960 1965Thr Leu
Gln His Arg Ile Ala Leu His Val Ala Asp Gly Leu Arg Tyr 1970 1975
1980Leu His Ser Ala Met Ile Ile Tyr Arg Asp Leu Lys Pro His Asn
Val1985 1990 1995 2000Leu Leu Phe Thr Leu Tyr Pro Asn Ala Ala Ile
Ile Ala Lys Ile Ala 2005 2010 2015Asp Tyr Xaa Ile Ala Gln Tyr Cys
Cys Arg Met Gly Ile Lys Thr Ser 2020 2025 2030Glu Gly Thr Pro Gly
Phe Arg Ala Pro Glu Val Ala Arg Gly Asn Val 2035 2040 2045Ile Tyr
Asn Gln Gln Ala Asp Val Tyr Ser Phe Gly Leu Leu Leu Tyr 2050 2055
2060Asp Ile Leu Thr Thr Gly Gly Arg Ile Val Glu Gly Leu Lys Phe
Pro2065 2070 2075 2080Asn Glu Phe Asp Glu Leu Glu Ile Gln Gly Lys
Leu Pro Asp Pro Val 2085 2090 2095Lys Glu Tyr Gly Cys Ala Pro Trp
Pro Met Val Glu Lys Leu Ile Lys 2100 2105 2110Gln Cys Leu Lys Glu
Asn Pro Gln Glu Arg Pro Thr Ser Ala Gln Val 2115 2120 2125Phe Asp
Ile Leu Asn Ser Ala Glu Leu Val Cys Leu Thr Arg Arg Ile 2130 2135
2140Leu Leu Pro Lys Asn Val Ile Val Glu Cys Met Val Ala Thr His
His2145 2150 2155 2160Asn Ser Arg Asn Ala Ser Ile Trp Leu Gly Cys
Gly His Thr Asp Arg 2165 2170 2175Gly Gln Leu Ser Phe Leu Asp Leu
Asn Thr Glu Gly Tyr Thr Ser Glu 2180 2185 2190Glu Val Ala Asp Ser
Arg Ile Leu Cys Leu Ala Leu Val His Leu Pro 2195 2200 2205Val Glu
Lys Glu Ser Trp Ile Val Ser Gly Thr Gln Ser Gly Thr Leu 2210 2215
2220Leu Val Ile Asn Thr Glu Asp Gly Lys Lys Arg His Thr Leu Glu
Lys2225 2230 2235 2240Met Thr Asp Ser Val Thr Cys Leu Tyr Cys Asn
Ser Phe Ser Lys Gln 2245 2250 2255Ser Lys Gln Lys Asn Phe Leu Leu
Val Gly Thr Ala Asp Gly Lys Leu 2260 2265 2270Ala Ile Phe Glu Asp
Lys Thr Val Lys Leu Lys Gly Ala Ala Pro Leu 2275 2280 2285Lys Ile
Leu Asn Ile Gly Asn Val Ser Thr Pro Leu Met Cys Leu Ser 2290 2295
2300Glu Ser Thr Asn Ser Thr Glu Arg Asn Val Met Trp Gly Gly Cys
Gly2305 2310 2315 2320Thr Lys Ile Phe Ser Phe Ser Asn Asp Phe Thr
Ile Gln Lys Leu Ile 2325 2330 2335Glu Thr Arg Thr Ser Gln Leu Phe
Ser Tyr Ala Ala Phe Ser Asp Ser 2340 2345 2350Asn Ile Ile Thr Val
Val Val Asp Thr Ala Leu Tyr Ile Ala Lys Gln 2355 2360 2365Asn Ser
Pro Val Val Glu Val Trp Asp Lys Lys Thr Glu Lys Leu Cys 2370 2375
2380Gly Leu Ile Asp Cys Val His Phe Leu Arg Glu Val Met Val Lys
Glu2385 2390 2395 2400Asn Lys Glu Ser Lys His Lys Met Ser Tyr Ser
Gly Arg Val Lys Thr 2405 2410 2415Leu Cys Leu Gln Lys Asn Thr Ala
Leu Trp Ile Gly Thr Gly Gly Gly 2420 2425 2430His Ile Leu Leu Leu
Asp Leu Ser Thr Arg Arg Leu Ile Arg Val Ile 2435 2440 2445Tyr Asn
Phe Cys Asn Ser Val Arg Val Met Met Thr Ala Gln Leu Gly
2450 2455 2460Ser Leu Lys Asn Val Met Leu Val Leu Gly Tyr Asn Arg
Lys Asn Thr2465 2470 2475 2480Glu Gly Thr Gln Lys Gln Lys Glu Ile
Gln Ser Cys Leu Thr Val Trp 2485 2490 2495Asp Ile Asn Leu Pro His
Glu Val Gln Asn Leu Glu Lys His Ile Glu 2500 2505 2510Val Arg Lys
Glu Leu Ala Glu Lys Met Arg Arg Thr Ser Val Glu 2515 2520
252527584DNAHomo sapiensmisc_feature149, 3364, 4321, 5096, 5457,
6055n = A,T,C or G 2atggctagtg gcagctgtca ggggtgcgaa gaggacgagg
aaactctgaa gaagttgata 60gtcaggctga acaatgtcca ggaaggaaaa cagatagaaa
cgctggtcca aatcctggag 120gatctgctgg tgttcacgta ctccgagcnc
gcctccaagt tatttcaagg caaaaatatc 180catgtgcctc tgttgatcgt
cttggactcc tatatgagag tcgcgagtgt gcagcaggtg 240ggttggtcac
ttctgtgcaa attaatagaa gtctgtccag gtacaatgca aagcttaatg
300ggaccccagg atgttggaaa tgattgggaa gtccttggtg ttcaccaatt
gattcttaaa 360atgctaacag ttcataatgc cagtgtaaac ttgtcagtga
ttggactgaa gaccttagat 420ctcctcctaa cttcaggtaa aatcaccttg
ctgatactgg atgaagaaag tgatattttc 480atgttaattt ttgatgccat
gcactcattt ccagccaatg atgaagtcca gaaacttgga 540tgcaaagctt
tacatgtgct gtttgagaga gtctcagagg agcaactgac tgaatttgtt
600gagaacaaag attatatgat attgttaagt gcgtcaacaa attttaaaga
tgaagaggaa 660attgtgcttc atgtgctgca ttgtttacat tccctagcga
ttccttgcaa taatgtggaa 720gtcctcatga gtggcaatgt caggtgttat
aatattgtgg tggaagctat gaaagcattc 780cctatgagtg aaagaattca
agaagtgagt tgctgtttgc tccataggct tacattaggt 840aattttttca
atatcctggt attaaacgaa gtccatgagt ttgtggtgaa agctgtgcag
900cagtacccag agaatgcagc attgcagatc tcagcgctca gctgtttggc
cctcctcact 960gagactattt tcttaaatca agatttagag gaaaagaatg
agaatcaaga gaatgatgat 1020gagggggaag aagataaatt gttttggctg
gaagcctgtt acaaagcatt aacgtggcat 1080agaaagaaca agcacgtgca
ggaggccgca tgctgggcac taaataatct ccttatgtac 1140caaaacagtt
tacatgagaa gattggagat gaagatggcc atttcccagc tcatagggaa
1200gtgatgctct ccatgctgat gcattcttca tcaaaggaag ttttccaggc
atctgcgaat 1260gcattgtcaa ctctcttaga acaaaatgtt aatttcagaa
aaatactgtt atcaaaagga 1320atacacctga atgttttgga gttaatgcag
aagcatatac attctcctga agtggctgaa 1380agtggctgta aaatgctaaa
tcatcttttt gaaggaagca acacttccct ggatataatg 1440gcagcagtgg
tccccaaaat actaacagtt atgaaacgtc atgagacatc attaccagtg
1500cagctggagg cgcttcgagc tattttacat tttatagtgc ctggcatgcc
agaagaatcc 1560agggaggata cagaatttca tcataagcta aatatggtta
aaaaacagtg tttcaagaat 1620gatattcaca aactggtcct agcagctttg
aacaggttca ttggaaatcc tgggattcag 1680aaatgtggat taaaagtaat
ttcttctatt gtacattttc ctgatgcatt agagatgtta 1740tccctggaag
gtgctatgga ttcagtgctt cacacactgc agatgtatcc agatgaccaa
1800gaaattcagt gtctgggttt aagtcttata ggatacttga ttacaaagaa
gaatgtgttc 1860ataggaactg gacatctgct ggcaaaaatt ctggtttcca
gcttataccg atttaaggat 1920gttgctgaaa tacagactaa aggatttcag
acaatcttag caatcctcaa attgtcagca 1980tctttttcta agctgctggt
gcatcattca tttgacttag taatattcca tcaaatgtct 2040tccaatatca
tggaacaaaa ggatcaacag tttctaaacc tctgttgcaa gtgttttgca
2100aaagtagcta tggatgatta cttaaaaaat gtgatgctag agagagcgtg
tgatcagaat 2160aacagcatca tggttgaatg cttgcttcta ttgggagcag
atgccaatca agcaaaggag 2220ggatcttctt taatttgtca ggtatgtgag
aaagagagca gtcccaaatt ggtggaactc 2280ttactgaata gtggatctcg
tgaacaagat gtacgaaaag cgttgacgat aagcattggg 2340aaaggtgaca
gccagatcat cagcttgctc ttaaggaggc tggccctgga tgtggccaac
2400aatagcattt gccttggagg attttgtata ggaaaagttg aaccttcttg
gcttggtcct 2460ttatttccag ataagacttc taatttaagg aaacaaacaa
atatagcatc tacactagca 2520agaatggtga tcagatatca gatgaaaagt
gctgtggaag aaggaacagc ctcaggcagc 2580gatggaaatt tttctgaaga
tgtgctgtct aaatttgatg aatggacctt tattcctgac 2640tcttctatgg
acagtgtgtt tgctcaaagt gatgacctgg atagtgaagg aagtgaaggc
2700tcatttcttg tgaaaaagaa atctaattca attagtgtag gagaatttta
ccgagatgcc 2760gtattacagc gttgctcacc aaatttgcaa agacattcca
attccttggg gcccattttt 2820gatcatgaag atttactgaa gcgaaaaaga
aaaatactat cttcagatga ttcactcagg 2880tcatcaaaac ttcaatccca
tatgaggcat tcagacagca tttcttctct ggcttctgag 2940agagaatata
ttacatcact agacctttca gcaaatgaac taagagatat tgatgcccta
3000agccagaaat gctgtataag tgttcatttg gagcatcttg aaaagctgga
gcttcaccag 3060aatgcactca cgagctttcc acaacagcta tgtgaaactc
tgaagagttt gacacatttg 3120gacttgcaca gtaataaatt tacatcattt
ccttcttatt tgttgaaaat gagttgtatt 3180gctaatcttg atgtctctcg
aaatgacatt ggaccctcag tggttttaga tcctacagtg 3240aaatgtccaa
ctctgaaaca gtttaacctg tcatataacc agctgtcttt tgtacctgag
3300aacctcactg atgtggtaga gaaactggag cagctcattt tagaaggaaa
taaaatatca 3360gggntatgct cccccttgag actgaaggaa ctgaagattt
taaaccttag taagaaccac 3420atttcatccc tatcagagaa ctttcttgag
gcttgtccta aagtggagag tttcagtgcc 3480agaatgaatt ttcttgctgc
tatgcctttc ttgcctcctt ctatgacaat cctaaaatta 3540tctcagaaca
aattttcctg tattccagaa gcaattttaa atcttccaca cttgcggtct
3600ttagatatga gcagcaatga tattcagtac ctaccaggtc ccgcacactg
gaaatctttg 3660aacttaaggg aactcttatt tagccataat cagatcagca
tcttggactt gagtgaaaaa 3720gcatatttat ggtctagagt agagaaactg
catctttctc acaataaact gaaagagatt 3780cctcctgaga ttggctgtct
tgaaaatctg acatctctgg atgtcagtta caacttggaa 3840ctaagatcct
ttcccaatga aatggggaaa ttaagcaaaa tatgggatct tcctttggat
3900gaactgcatc ttaactttga ttttaaacat ataggatgta aagccaaaga
catcataagg 3960tttcttcaac agcgattaaa aaaggctgtg ccttataacc
gaatgaaact tatgattgtg 4020ggaaatactg ggagtggtaa aaccacctta
ttgcagcaat taatgaaaac caagaaatca 4080gatcttggaa tgcaaagtgc
cacagttggc atagatgtga aagactggcc tatccaaata 4140agagacaaaa
gaaagagaga tctcgtccta aatgtgtggg attttgcagg tcgtgaggaa
4200ttctatagta ctcatcccca ttttatgacg cagcgagcat tgtaccttgc
tgtctatgac 4260ctcagcaagg gacaggctga agttgatgcc atgaagcctt
ggctcttcaa tataaaggct 4320ngcgcttctt cttcccctgt gattctcgtt
ggcacacatt tggatgtttc tgatgagaag 4380caacgcaaag cctgcatgag
taaaatcacc aaggaactcc tgaataagcg agggttccct 4440gccatacgag
attaccactt tgtgaatgcc accgaggaat ctgatgcttt ggcaaaactt
4500cggaaaacca tcataaacga gagccttaat ttcaagatcc gagatcagct
tgttgttgga 4560cagctgattc cagactgcta tgtagaactt gaaaaaatca
ttttatcgga gcgtaaaaat 4620gtgccaattg aatttcccgt aattgaccgg
aaacgattat tacaactagt gagagaaaat 4680cagctgcagt tagatgaaaa
tgagcttcct cacgcagttc actttctaaa tgaatcagga 4740gtccttcttc
attttcaaga cccagcactg cagttaagtg acttgtactt tgtggaaccc
4800aagtggcttt gtaaaatcat ggcacagatt ttgacagtga aagtggaagg
ttgtccaaaa 4860caccctaagg gcattatttc gcgtagagat gtggaaaaat
ttctttcaaa aaaaaggaaa 4920tttccaaaga actacatgtc acagtatttt
aagctcctag aaaaattcca gattgctttg 4980ccaataggag aagaatattt
gctggttcca agcagtttgt ctgaccacag gcctgtgata 5040gagcttcccc
attgtgagaa ctctgaaatt atcatccgac tatatgaaat gccttntttt
5100ccaatgggat tttggtcaag attaatcaat cgattacttg agatttcacc
ttacatgctt 5160tcagggagag aacgagcact tcgcccaaac agaatgtatt
ggcgacaagg catttactta 5220aattggtctc ctgaagctta ttgtctggta
ggatctgaag tcttagacaa tcatccagag 5280agtttcttaa aaattacagt
tccttcttgt agaaaaggct gtattctttt gggccaagtt 5340gtggaccaca
ttgattctct catggaagaa tggtttcctg ggttgctgga gattgatatt
5400tgtggtgaag gagaaactct gttgaagaaa tgggcattat atagttttaa
tgatggngaa 5460gaacatcaaa aaatcttact tgatgacttg atgaagaaag
cagaggaagg agatctctta 5520gtaaatccag atcaaccaag gctcaccatt
ccaatatctc agattgcccc tgacttgatt 5580ttggctgacc tgcctagaaa
tattatgttg aataatgatg agttggaatt tgaacaagct 5640ccagagtttc
tcctaggtga tggcagtttt ggatcagttt accgagcagc ctatgaagga
5700gaagaagtgg ctgtgaagat ttttaataaa catacatcac tcaggctgtt
aagacaagag 5760cttgtggtgc tttgccacct ccaccacccc agtttgatat
ctttgctggc agctgggatt 5820cgtccccgga tgttggtgat ggagttagcc
tccaagggtt ccttggatcg cctgcttcag 5880caggacaaag ccagcctcac
tagaacccta cagcacagga ttgcactcca cgtagctgat 5940ggtttgagat
acctccactc agccatgatt atataccgag acctgaaacc ccacaatgtg
6000ctgcttttca cactgtatcc caatgctgcc atcattgcaa agattgctga
ctacngcatt 6060gctcagtact gctgtagaat ggggataaaa acatcagagg
gcacaccagg gtttcgtgca 6120cctgaagttg ccagaggaaa tgtcatttat
aaccaacagg ctgatgttta ttcatttggt 6180ttactactct atgacatttt
gacaactgga ggtagaatag tagagggttt gaagtttcca 6240aatgagtttg
atgaattaga aatacaagga aaattacctg atccagttaa agaatatggt
6300tgtgccccat ggcctatggt tgagaaatta attaaacagt gtttgaaaga
aaatcctcaa 6360gaaaggccta cttctgccca ggtctttgac attttgaatt
cagctgaatt agtctgtctg 6420acgagacgca ttttattacc taaaaacgta
attgttgaat gcatggttgc tacacatcac 6480aacagcagga atgcaagcat
ttggctgggc tgtgggcaca ccgacagagg acagctctca 6540tttcttgact
taaatactga aggatacact tctgaggaag ttgctgatag tagaatattg
6600tgcttagcct tggtgcatct tcctgttgaa aaggaaagct ggattgtgtc
tgggacacag 6660tctggtactc tcctggtcat caataccgaa gatgggaaaa
agagacatac cctagaaaag 6720atgactgatt ctgtcacttg tttgtattgc
aattcctttt ccaagcaaag caaacaaaaa 6780aattttcttt tggttggaac
cgctgatggc aagttagcaa tttttgaaga taagactgtt 6840aagcttaaag
gagctgctcc tttgaagata ctaaatatag gaaatgtcag tactccattg
6900atgtgtttga gtgaatccac aaattcaacg gaaagaaatg taatgtgggg
aggatgtggc 6960acaaagattt tctccttttc taatgatttc accattcaga
aactcattga gacaagaaca 7020agccaactgt tttcttatgc agctttcagt
gattccaaca tcataacagt ggtggtagac 7080actgctctct atattgctaa
gcaaaatagc cctgttgtgg aagtgtggga taagaaaact 7140gaaaaactct
gtggactaat agactgcgtg cactttttaa gggaggtaat ggtaaaagaa
7200aacaaggaat caaaacacaa aatgtcttat tctgggagag tgaaaaccct
ctgccttcag 7260aagaacactg ctctttggat aggaactgga ggaggccata
ttttactcct ggatctttca 7320actcgtcgac ttatacgtgt aatttacaac
ttttgtaatt cggtcagagt catgatgaca 7380gcacagctag gaagccttaa
aaatgtcatg ctggtattgg gctacaaccg gaaaaatact 7440gaaggtacac
aaaagcagaa agagatacaa tcttgcttga ccgtttggga catcaatctt
7500ccacatgaag tgcaaaattt agaaaaacac attgaagtga gaaaagaatt
agctgaaaaa 7560atgagacgaa catctgttga gtaa 7584322DNAArtificial
SequencePrimer 3ttgcagctgt aaggaatttg gg 22421DNAArtificial
SequencePrimer 4gcattcttca gcctgagacc c 21522DNAArtificial
SequencePrimer 5tgaaggacac tgaacaagat gg 22622DNAArtificial
SequencePrimer 6gccatagtcc ttccatagtt cc 22718DNAArtificial
SequencePrimer 7cgcagcgagc attgtacc 18820DNAArtificial
SequencePrimer 8ctcggaaagt ttcccaattc 20924DNAArtificial
SequencePrimer 9ctggtattac ctcaactgtg gctc 241023DNAArtificial
SequencePrimer 10actggtatgt ttaagcctgg cac 231124DNAArtificial
SequencePrimer 11agcagcagag aagatttcaa taac 241222DNAArtificial
SequencePrimer 12aatcatcttt gaaagaacca gg 221324DNAArtificial
SequencePrimer 13taaacgaagc tccctcactg taag 241422DNAArtificial
SequencePrimer 14tctttgtagc tgcggttgtt tc 221524DNAArtificial
SequencePrimer 15tcatgaagat gtctgtgata gggc 241624DNAArtificial
SequencePrimer 16ctctattgtg agcaaactgc atgg 241726PRTHomo sapiens
17Asp Tyr Gly Ile Ala Gln Tyr Cys Cys Arg Met Gly Ile Lys Thr Ser1
5 10 15Glu Gly Thr Pro Gly Phe Arg Ala Pro Glu 20 251826PRTHomo
sapiens 18Asp Tyr Gly Ile Ser Arg Gln Ser Phe His Glu Gly Ala Leu
Gly Val1 5 10 15Glu Gly Thr Pro Gly Tyr Gln Ala Pro Glu 20
251925PRTHomo sapiens 19Asp Phe Gly Leu Ala Lys Ala Glu Arg Lys Gly
Leu Asp Ser Ser Arg1 5 10 15Leu Pro Val Lys Trp Thr Ala Pro Glu 20
252030PRTHomo sapiens 20Asp Phe Gly Leu Ala Arg Asp Ile Met His Asp
Ser Asn Tyr Val Ser1 5 10 15Lys Gly Ser Thr Phe Leu Pro Val Lys Trp
Met Ala Pro Glu 20 25 302126PRTHomo sapiens 21Asp Phe Gly Leu Ala
Arg Glu Trp His Lys Thr Thr Lys Met Ser Ala1 5 10 15Ala Gly Thr Tyr
Ala Trp Met Ala Pro Glu 20 252219PRTHomo sapiens 22Asp Phe Gly Asn
Glu Phe Lys Asn Ile Phe Gly Thr Pro Glu Phe Val1 5 10 15Ala Pro
Glu2330PRTHomo sapiens 23Asp Phe Gly Leu Ala Thr Val Lys Ser Arg
Trp Ser Gly Ser His Gln1 5 10 15Phe Glu Gln Leu Ser Gly Ser Ile Leu
Trp Met Ala Pro Glu 20 25 302417PRTHomo sapiens 24Ile Ala Lys Ile
Ala Asp Tyr Gly Ile Ala Gln Tyr Cys Cys Arg Met1 5 10
15Gly2517PRTTetraodon nigroviridis 25Ile Ala Lys Ile Thr Asp Tyr
Gly Ile Ala Gln His Cys Cys Ser Met1 5 10 15Gly
* * * * *